This invention relates to novel helminth parasite antigens and their use in the control of disease caused by helminth parasites, particularly parasitic nematodes of the gastro-intestinal tract of mammals.
Helminth parasites, particularly nematodes, infect or infest a wide range of animals, including man, and are a widespread and significant source of disease and ill-thrift, not only in animals, but also in man. Such parasites thus represent a considerable worldwide drain on economic resources. This is particularly true in animal husbandry, where parasite infections of grazing animals, such as sheep and cattle, are often difficult and expensive to control and may result in significant economic losses.
Particular mention may be made in this regard of the non-blood feeding nematodes Ostertagia ostertagi and Ostertagia (Teladorsagia) circumcincta (O. circumcincta has recently been reclassified as T.circumcincta, although the new name is not yet in wide usage).
Other parasitic helminths of economic importance include the various species of the following helminth families: Trichostrongylus, Nematodirus, Dictyocaulus, Cooperia, Trichuris, Oesophagostomum, Bunostomum and Metastrongylus. In addition to domestic livestock, pets and humans may also be infected, not infrequently with fatal results.
At present, control of helminth parasites of grazing livestock relies primarily on the use of anthelmintic drugs, combined with pasture management. Such techniques have not proved entirely satisfactory however, due to their expense and inconvenience and to a rapid increase in drug resistance. Anthelmintic drugs need to be administered frequently and appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
To overcome these problems, attempts have been made to achieve immunological means of control. Although there has been some success in identifying certain protective antigens as potential vaccine candidates, most notably in Haemonchus, this approach has proved difficult and, other than for the cattle lungworm Dictyocaulus viviparus, has yet to come to commercial fruition.
The most success to date has been achieved with the protein doublet H110D, an integral membrane protein isolated from the gut of H.contortus and described by Munn in WO88/00835. H110D now represents the most promising vaccine candidate to date.
Munn has also described and proposed as a vaccine, contortin, a helical polymeric extracellular protein associated with the luminal surface of H.contortus intestinal cells (Munn et al., Parasitology 94: 385-397, 1987).
A third Haemonchus gut membrane protein with protective antigenic properties has also been discovered and termed H45 (Munn and Smith, WO90/11086).
Whilst proteins such as H110 D and H45 can be used as the basis for a vaccine against Haemonchus, there is nonetheless a continuing need for new and improved helminth parasite vaccines and in particular for a vaccine which may be used across a broad range of helminth genera. Most studies to date have concentrated on blood feeding nematodes such as Haemonchus and there is especially a need for vaccines against non-blood feeding helminths such as Trichostrongylus, Cooperia, Dictyocaulus, and particularly Ostertagia.
The present invention accordingly seeks to provide novel antigens for use as helminth parasite vaccines and in particular as protective immunogens in the control of diseases caused by non-blood feeding helminth parasites.
More specifically, the present invention is based on the finding that the gut of non-blood feeding nematodes such as Ostertagia and Cooperia contains an integral membrane protein having aminopeptidase activity which is believed to be an important protective antigen capable of conferring protective immunity against helminth parasites in animals. Such proteins, when liberated from the membranes in which they are integral, for example by the use of detergents, are novel and of use in the manufacture of vaccines against helminth infections.
According to one aspect, the present invention thus provides a protective helminth parasite antigen which is characterised by possessing aminopeptidase-like activity and which, in native form, is an integral membrane protein associated with the gut microvilli of a non-obligate blood feeding helminth parasite, or a functionally-equivalent variant, or antigenic fragment or precursor thereof.
A further aspect of the invention provides such protective antigens, and functionally-equivalent variants, antigenic fragments or precursors thereof, for use in stimulating an immune response against helminth parasites in a human or non-human, preferably mammalian, especially preferably ruminant, animal.
A precursor for the antigen in question may be a larger protein which is processed, eg. by proteolysis, to yield the antigen per se. Such precursors may take the form of zymogens ie. inactive precursors of enzymes, activated by proteolytic cleavage, for example analogous to the pepsin/pepsinogen system or the well known zymogens involved in the blood clotting cascade.
Non-obligate blood feeding parasites are defined for present purposes as those which imbibe host blood rarely and seemingly incidentally (in contrast to obligate blood feeders such as Haemonchus, which feed almost exclusively on host blood) and include both plug feeders and browsers. Plug-feeding worms have enlarged buccal capsules which they use to envelope a plug of host tissue e.g. the intestinal mucosa in the case of Chabertia ovina) from which they obtain nutrient. Browsing nematodes do not have mouthparts specialised for attachment. The composition of their diet is not known but is thought to consist either of host tissue, extracellular tissue fluid, mucus or, in the case of gastro-intestinal species, host digesta. Some gastrointestinal species, particularly those which live in close association with the mucosa (e.g. Osteragia circumcincta in the abomasal glands and certain Trichostrongylus species (e.g. other than T.axei) which tunnel under the intestinal epithelium may feed on a mixture of these.
Economically important helminth parasites which are not obligate blood feeders include the following genera and their subspecies: Ostertagia (which, for the avoidance of doubt, as used herein includes Teladorsagia), Trichostrongylus spp. (e.g. T.colubriformis, T.vitrinus and T.axei), Cooperia spp., Nematodirus, Chabertia and Oesophagostomum.
The novel antigens of the invention are not recognised by sera from naturally immune animals. In other words, they are not normally, in native form, accessible to the immune system of the infected host and are thus xe2x80x9chiddenxe2x80x9d, xe2x80x9cconcealedxe2x80x9d or xe2x80x9ccrypticxe2x80x9d antigens.
The term xe2x80x9cprotective antigensxe2x80x9d or xe2x80x9cprotective antigenic activityxe2x80x9d as used herein defines those antigens and their fragments or precursors, capable of generating a host-protective, ie. immunogenic, immune response, that is a response by the host which leads to generation of immune effector molecules, antibodies or cells which damage, inhibit or kill the parasite and thereby xe2x80x9cprotectxe2x80x9d the host from clinical or sub-clinical disease and loss of productivity. Such a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death.
As mentioned above, included within the scope of the invention are functionally-equivalent variants of the novel antigens and their fragments and precursors. xe2x80x9cFunctionally-equivalentxe2x80x9d is used herein to define proteins related to or derived from the native protein, where the amino acid sequence has been modified by single or multiple amino acid substitution, addition and/or deletion and also sequences where the amino acids have been chemically modified, including by deglycosylation or glycosylation, but which nonetheless retain protective antigenic activity eg. are capable of raising host protective antibodies and/or functional immunity against the parasites. Within the meaning of xe2x80x9cadditionxe2x80x9d variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide fused to the aminopeptidase antigen sequence. Such functionally-equivalent variants mentioned above include natural biological variations (eg. allelic variants or geographical variations within a species) and derivatives prepared using known techniques. For example, functionally-equivalent proteins may be prepared either by chemical peptide synthesis or in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids. Functionally-equivalent variants according to the invention also include analogues in different parasite genera or species.
Aminopeptidase enzymes are believed to function as part of the nutrient assimilation process of helminths and blockage of enzymic activity may contribute to the protective effect of the antibodies induced in response to immunisation with the antigens of the invention. Aminopeptidase-like activity may be assayed using a range of substrates, for example alanine, leucine and xcex1-glutamyl-p-nitroanilide.
Antigens of the invention have been shown to possess a range of aminopeptidase activities, most notably aminopeptidase M(ApM)-like and aminopeptidase A (ApA)-like activity, which may be demonstrated using e.g. alanine, leucine or methionine p-nitroanilide and xcex1-glutamic acid p-nitroanilide respectively, for example in helminth extracts or on substrate-gels following polyacrylamide-gel electophoresis (PAGE) using corresponding xcex2-methoxy naphthylamide substrates. In addition, inhibitor studies have shown the antigens to be metalloproteinases.
The aminopeptidase antigens of the invention are integral membrane proteins, as may be shown by detergent solubilization studies. For example, the antigens are insoluble in aqueous buffers, such as phosphate-buffered saline (PBS) and detergents such as polyoxyethylene-sorbitan-monolaurate (Tween) which solubilise membrane-associated proteins, but may be solubilised by non-ionic detergents, e.g. TRITON X-100, or dodecylpolyethylene glycolether (THESIT, from Desitin Werk, Hamburg, Germany) which solubilise integral membrane proteins.
Histochemical studies have shown the antigens of the invention to be associated with the intestinal microvilli of the parasites. Thus, for example in the non-obligate blood feeding helminths e.g. Ostertagia and Cooperia, staining of frozen sections reveals the presence of aminopeptidase activity in the area of the intestinal microvilli.
Lectin binding studies have further shown that the antigens of the invention bind to concanavalin A (ConA), indicating the presence of glycosylation, particularly mannose and/or fucose residues.
Preferred antigens according to the invention are those isolatable from Ostertagia. In particular, in both O.circumcincta and O.ostertagia aminopeptidase activity (including ApA- and ApM-like) has been shown to be associated with a band of material which may be separated by electrophoresis on polyacrylamide gel under denaturing conditions, and which has an apparent molecular weight (Mr) of about 124,000-126,000 (6-16% SDS-PAGE). Such proteins and their analogues in other non-blood feeders represent particularly preferred embodiments of the invention.
Antigens according to the invention may be obtained by extraction of non-obligate blood feeding helminth parasites using appropriate detergents.
A further aspect of the invention thus provides a method for preparing the above-mentioned antigens of the invention which comprises the step of subjecting a crude extract of a non-obligate blood feeding helminth parasite to detergent extraction, eg. using Tween 20 or a like detergent to remove membrane-associated proteins followed by Thesit or a like detergent, and recovering the solubilised integral membrane proteins.
The detergent extract may then be subjected to further purification using conventional procedures eg. centrifugation, selective precipitation, electrophoresis, chromatography and the like. Fractions containing the antigen of the invention may be identified by assaying for aminopeptidase activity using known techniques.
The antigen may be released from the membrane in a truncated form, which is soluble in water without addition of detergent, by treatment with a proteolytic enzyme such as trypsin.
Antigens obtainable by such methods form a further aspect of the invention.
The crude extract of the helminth parasite may be prepared using conventional biochemical and surgical techniques eg. by homogenisation of the whole or a portion of the parasite. Thus, for example the parasites may be subjected to homogenisation in a suitable buffer or medium such as phosphate buffered saline (PBS), and the insoluble material (ie. the pellet) may be recovered by centrifugation, whereby to form the required crude extract.
Thus, a suitable purification protocol for the antigens of the invention might comprise (i) homogenising adult parasite worms in PBS, recovering the PBS-insoluble material (eg. by centrifugation), optionally repeating the PBS-washing step one or more times, (ii) extracting the PBS-insoluble material (ie. the pellet) with aqueous Tween 20 (eg. in PBS), (iii) recovering the Tween-insoluble fraction e.g. by centrifugation, (iv) extracting the Tween-insoluble material with aqueous Thesit (e.g. in PBS), (v) recovering the solubilised fraction containing the Thesit-soluble proteins, (ie. the supernatent also known as ThHS) followed by (vi) ion exchange chromatography eg. on a Mono Q column, and/or gel filtration eg. using Superose. A ConA affinity chromatography step may optionally also be included e.g. before ion exchange.
An alternative preparation process may take advantage of the fact that the target antigens have particular selective binding activities by using an affinity chromatography system in which specific ligands are immobilised on a solid phase matrix.
Thus the invention also provides a process for preparing the above-mentioned antigens of the invention, said process comprising preparing an extract of said parasite containing at least one protective antigen as defined above, purifying said antigen therefrom by binding said antigen to an immobilized phase including a specific binding partner for the antigen and subsequently eluting said antigen from said immobilized phase. Suitable specific binding partners include substrate analogues, for example the inhibitor bestatin, and oligosaccharide specific lectins e.g. ConA.
The invention also provides use of a helminth parasite antigen as hereinbefore defined, and fragments, precursors and functionally-equivalent variants thereof, for the preparation of a vaccine composition for use in stimulating an immune response against helminth parasites in a human or non-human, animal.
Further provided according to the invention is a vaccine composition for stimulating an immune response against helminth parasites in a human or non-human animal comprising one or more antigens, antigenic fragments, precursors or functionally-equivalent variants thereof, as defined above, together with a pharmaceutically acceptable carrier or diluent, and a method of stimulating an immune response against helminth parasites in a human or non-human animal, comprising administering to said animal a vaccine composition as defined above.
The animal preferably is mammalian and more preferably a ruminant.
As mentioned above, antigens according to the invention may be obtained from a range of non-obligate blood feeding helminth parasite genera. Preferably, however the helminths will be nematodes, especially preferably gastro-intestinal nematodes including for example Ostertagia and Cooperia. As mentioned above, antigens from Ostertagia are especially preferred. Such antigens may be used to prepare vaccines against a range of helminth parasites including any of those mentioned above. Preferred are those antigens, so called xe2x80x9cbroad spectrumxe2x80x9d antigens, which are capable of stimulating host protective immune responses against, in addition to the parasite from which they were isolated, a broad range of other parasites.
As mentioned above, one of the ways in which the antigens of the invention may exert their host protective effects is by raising inhibitory antibodies which inhibit the growth, maintenance and/or development of the parasite. Such antibodies and their antigen-binding fragments (eg. F(ab)2, Fab and Fv fragments ie. fragments of the xe2x80x9cvariablexe2x80x9d region of the antibody, which comprises the antigen binding site) which may be mono- or polyclonal, form a further aspect of the invention, as do vaccine compositions containing them and their use in the preparation of vaccine compositions for use in passively immunising hosts against parasites. Such inhibitory antibodies may be raised by use of idiotypic antibodies. Anti-idiotypic antibodies may be used as immunogens in vaccines.
In addition to the extraction and isolation techniques mentioned above, the antigens may be prepared by recombinant DNA technology using standard techniques, such as those described for example by Sambrook et al., 1989, (Molecular Cloning, a laboratory manual 2nd Edition, Cold Spring Harbor Press).
Nucleic acid molecules comprising a nucleotide sequence encoding the antigens of the invention thus form further aspects of the invention.
Nucleic acid molecules according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA, and include degenerate, substantially homologous and hybridising sequences which are capable of coding for the antigen or antigen fragment or precursor concerned. By xe2x80x9csubstantially homologousxe2x80x9d is meant sequences displaying at least 60%, preferably at least 70% or 80% sequence homology. Hybridising sequences included within the scope of the invention are those binding under non-stringent conditions (6xc3x97SSC/50% formamide at room temperature) and washed under conditions of low stringency (2xc3x97SSC, room temperature, more preferably 2xc3x97SCC, 42xc2x0 C.) or conditions of higher stringency eg. 2xc3x97SSC, 65xc2x0 C. (where SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2), as well as those which, but for the degeneracy of the code, would hybridise under the above-mentioned conditions.
Derivatives of nucleotide sequences capable of encoding antigenically active antigens or antigen variants according to the invention may be obtained by using conventional methods well known in the art.
Antigens according to the invention may be prepared in recombinant form by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly defined above, operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Synthetic polypeptides expressed in this manner form a further aspect of this invention (the term xe2x80x9cpolypeptidexe2x80x9d is used herein to include both full-length protein and shorter length peptide sequences).
The antigen so expressed may be a fusion polypeptide comprising all or a portion of an antigen according to the invention and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. This may for example be xcex2-galactosidase, glutathione-S-transferase, hepatitis core antigen or any of the other polypeptides commonly employed in fusion proteins in the art.
Other aspects of the invention thus include cloning and expression vectors containing the DNA coding for an antigen of the invention and methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the antigen into vector nucleic acid, eg. vector DNA. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codons, ribosomal binding sites) and transcriptional control elements (eg. promoter-operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art. Suitable viral vectors include baculovirus and also adenovirus and vaccinia viruses. Many other viral vectors are described in the art.
A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature.
The invention also includes transformed or transfected prokaryotic or eukaryotic host cells, or transgenic organisms containing a nucleic acid molecule according to the invention as defined above. Such host cells may for example include prokaryotic cells such as E.coli, eukaryotic cells such as yeasts or the baculovirus-insect cell system, transformed mammalian cells and transgenic animals and plants. Particular mention may be made of transgenic nematodes (see for example Fire, 1986, EMBO J., 5. 2673 for a discussion of a transgenic system for the nematode Caenorhabditis).
A further aspect of the invention provides a method for preparing an antigen of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule encoding all or a portion of said antigen, under conditions whereby said antigen is expressed and recovering said antigen thus produced.
The antigens of the invention and functionally equivalent antigen variants may also be prepared by chemical means, such as the well known Merrifield solid phase synthesis procedure.
Water soluble derivatives of the novel antigens discussed above form a further aspect of the invention. Such soluble forms may be obtained by proteolytic digestion, for example using the enzyme trypsin. Generally speaking enzymic digestion of the antigens yields two fractions, a detergent soluble fraction (which remains with the membrane) and a water-soluble fraction.
A vaccine composition may be prepared according to the invention by methods well known in the art of vaccine manufacture. Traditional vaccine formulations may comprise one or more antigens or antibodies according to the invention together, where appropriate, with one or more suitable adjuvants eg. aluminium hydroxide, saponin, quil A, or more purified forms thereof, muramyl dipeptide, mineral or vegetable oils, Novasomes or non-ionic block co-polymers or DEAE dextran, in the presence of one or more pharmaceutically acceptable carriers or diluents. Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or polypeptides into an animal or patient. Additional components such as preservatives may be included.
An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eg. vaccinia virus, adenovirus, bacteria such as the Bacillus Calmette-Guxc3xa9rin strain of Mycobacterium bovis (BCG) or Salmonella spp) which may be live, killed or attenuated, having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
Administration of the vaccine composition may take place by any of the conventional routes, eg. orally or parenterally such as by intramuscular injection, optionally at intervals eg. two injections at a 7-35 day interval.
The antigens may be used according to the invention in combination with other protective antigens obtained from the same or different parasite species. Such a combined vaccine composition may contain smaller amounts of the various antigens than an individual vaccine preparation, containing just the antigen in question. Combined vaccines are beneficial where there is a likelihood that xe2x80x9cadaptive selectionxe2x80x9d of the parasite may occur when a single antigen vaccine is used.
Animals which may benefit from the present invention may be any human or non-human animal, but companion animals, particularly dogs and cats and domestic animals, especially ruminants are preferred. Particular mention may be made of sheep, cattle, pigs, deer and goats.
Treatment of membrane preparations obtained from O. ostertagi with trypsin released aminopeptidase M activity in a water-soluble form which bound to ConA and after gel filtration yielded a fraction with high activity and showing a Coomassie blue staining band on SDS-PAGE around 116,000. The N-terminal sequence of a band of similar size obtained by SDS-PAGE of a breakdown product of 012 was Ala Glu Asp Leu Arg Leu Pro Thr Asn Ile Arg Pro Leu Ile Tyr Asp Leu Thr (AEDLRLPTNIRPLIYDLT, SEQ ID NO:1). This shows 67% identity and 89% similarity to the sequence of the corresponding region of microsomal aminopeptidases H110D-1, -2 and -3 from Haemonchus contortus deduced from their DNA sequences and 67% identity to the observed N-terminal amino acid sequence of a water soluble fragment released from H110D by trypsin cleavage. The amino acids Leu, Pro, Pro and Tyr at positions 6, 7, 12 and 15 respectively of this peptide are conserved in human, pig, rat and mouse microsomal aminopeptidases.
The full sequence of the O12 microsomal aminopeptidase was obtained by sequencing cloned PCR products generated from Ostertagia ostertagi first strand cDNA and primers based on a) the sequence GAMENWGL (SEQ ID NO: 2) common to the H110D microsomal aminopeptidases, b) at the 5xe2x80x2 end, the helminth spliced leader sequence SL1 and c) a vector sequence at the 3xe2x80x2 end.
Antibodies to O12 do not form in the course of infection but the protein is shown to be highly immunogenic in sheep and cattle when injected in purified form. Antibodies raised against O12 from O. ostertagi cross-react with periodate-treated O12 from O. circumcincta as do antibodies raised in sheep to CamQH110D.
This invention will now be described in more detail with particular reference to the protein O12 from Ostertagia ostertagi. However, by a variety of techniques such as histochemistry and Western blotting, O12 equivalents have been demonstrated in the parasites Ostertagia circumcincta and Cooperia oncophora. It is believed that the O12 protein is a multigene complex and that in addition, the nucleotide sequences encoding it, may exhibit sequence variations between different strains and different life cycle stages of the helminth. Moreover there may exist multiple enzyme forms (isoenzymes) which may be differentially expressed at different stages, or in different strains. In this study DNA sequences, and thus the predicted amino acid sequences, have been determined from cDNA clones and PCR products obtained from mRNA corresponding to the O12 gene by recombinant DNA technology from 12-days old (early fifth stage) Ostertagia ostertagi. 
Sequencing of PCR products has enabled us to identify closely related O12 sequences which are here designated O12-1 (SEQ ID NO:19), O12-2 (SEQ ID NO:20), O12-3 (SEQ ID NO:21), O12-4 (SEQ ID NO:22) and O12-5 (SEQ ID NO:23). O12-1 and O12-2 are 5xe2x80x2 sequences of about 1 kb terminating at their 3xe2x80x2 ends in the primer based on the conserved sequence GAMENWGL (SEQ ID NO:2); O12-3, O12-4 and O12-5are 3xe2x80x2 sequences of about 2.5 kb originating at their 5xe2x80x2 ends with this primer.
The relationships of O12-1 and -2 with O12-3, -4 and -5 may be determined by PCRing between primers towards the 3xe2x80x2 ends of O12-1 and -2 and the 5xe2x80x2 ends of O12-3, -4 and -5 to include the common GAMENWGL (SEQ ID NO:2) sequence.
Differences and variations in the sequences obtained from the PCR products have been observed, as can be seen in particular from FIGS. 19 and 20 (SEQ ID NO:23) and as summarised below.
Homologies of the deduced amino acid sequences (SEQ ID NO:24-28) obtained by translation of the nucleotide sequences shown in FIGS. 19 and 20 (SEQ ID NO:19-23).
The differences can be attributed to different mRNAs multigene family.
Comparison of the sequences of various of the clones mentioned above, against computer databases of known sequences, reveals substantial homology with the family of microsomal aminopeptidase enzymes (EC. 3.4.11.-). Enzymological activity and inhibitor studies performed with the O12 protein and subfractions thereof confirm that the protein is in fact microsomal aminopeptidase (xcex1-amino acyl peptide hydrolase (microsomal)). Using the enzyme trypsin, it was found that O12 may be partially cleaved, forming two fractions, a detergent-soluble fraction (which remained with the membrane) and a water-soluble fraction (which is designated O12S); it was found that aminopeptidase M-like activity is associated with the water-soluble O12S fraction.
An eighteen amino acid N-terminal sequence (designated Pep A, SEQ ID NO:1) from a proteolytic fragment of O12 has 67% identity and 89% similarity to the corresponding fragments of H110D-1, -2 and -3 deduced from their DNA sequences. The sequences are
PepA: AEDLRLPTNIRPLIYDLT (SEQ ID NO:1)
H11-1: AEELRLPTTIKPLTYDLV (SEQ ID NO:29)
PepA: AEDLRLPTNIRPLIYDLT (SEQ ID NO:1)
H11-2: AEELLLPTNIKPVSYDLN (SEQ ID NO:30)
PepA: AEDLRLPTNIRPLIYDLT (SEQ ID NO:15)
H11-3: AAELLLPSNIKPLSYDLT (SEQ ID NO:16)
Over this region the three deduced H11 sequences are 72-77% identical (13 or 14 amino acids) and 83-100% similar.) Nine amino acids are conserved in all four sequences:
A12L3LP45I6P7XYDLX
For the other residues, 1 is either E or A, 2 is D in PepA and E in H11, 3 is L or R, 5 is N or T, 6 is R in PepA and K in E11, and 7 is L or V (LP P Y are conserved in human, pig, rat and mouse microsomal aminopeptidases.)
Comparing the amino acid sequences of PepA (SEQ ID NO:1) and H110D (H11) as determined we have:
PepA ARDLRLPTNIRPLIYDLT (SEQ ID NO:1) (N-terminus of a breakdown product.)
H11 AEELLLPTNIKPVSYDLK (SEQ ID NO:32) (N-terminus of a trypsin fragment.) this has 67% identity and 83% similarity.
PCR from O. ostertagi first strand cDNA using an oligonucleotide corresponding to the H110D conserved sequence GAMENWGL (SEQ ID NO:2) has yielded products of the expected sizes, about 1 kb with the 5xe2x80x2 primer and about 2.5 kb with the 3xe2x80x2 primer. Two closely related but distinguishable 5xe2x80x2 sequences designated O12-1 and O12-2 have been obtained (see FIG. 19, SEQ ID NOS: 19 AND 20). Over the region above, the deduced sequences for O12-1 and O12-2 are 72% identical.
O12-1 AEELRLPSVIPPLLYDLS (SEQ ID NO:41)
O12-2 AEBLRLPTNIKPLLYNLT (SEQ ID NO:42)
PepA AEDLRLPTNIRPLIYDLT (SEQ ID NO:1)
O12-1 AEELRLPSVIPPLLYDLS (SEQ ID NO:41)
There is 72% identity of the determined amino acid sequence of PepA (SEQ ID NO:1) with the sequence deduced from clone O12-1 and 67% identity with the sequence from clone O12-2.